Entomopathogenic nematode detection and counting model developed based on A-star algorithm.

Entomopathogenic nematodes are soil-dwelling living organisms widely employed in the biological control of agricultural insect pests, serving as a significant alternative to pesticides. In laboratory procedures, the counting process remains the most common, labor-intensive, time-consuming, and approximate aspect of studies related to entomopathogenic nematodes. In this context, a novel method has been proposed for the detection and quantification of Steinernema feltiae isolate using computer vision on microscope images. The proposed method involves two primary algorithmic steps: framing and isolation. Compared to YOLO-V5m, YOLO-V7m, and YOLO-V8m, the A-star-based developed network demonstrates significantly improved detection accuracy compared to other networks. The novel method is particularly effective in facilitating the detection of overlapping nematodes. The developed algorithm excludes processes that increase space and time complexity, such as the weight document, which contains the learned parameters of the deep learning model, model integration, and prediction time, resulting in more efficient operation. The results indicate the feasibility of the proposed method for detecting and counting entomopathogenic nematodes.

Toxicological impacts of microplastics on virulence, reproduction and physiological process of entomopathogenic nematodes.

Microplastics have emerged as significant and concerning pollutants within soil ecosystems. Among the soil biota, entomopathogenic nematodes (EPNs) are lethal parasites of arthropods, and are considered among the most effective biological agents against pests. Infective juveniles (IJs) of EPNs, as they navigate the soil matrix scavenging for arthropod hosts to infect, they could potentially encounter microplastics. Howver, the impact of microplastics on EPNs has not been fully elucidated yet. We addressed this gap by subjecting Steinernema feltiae EPNs to polystyrene microplastics (PS-MPs) with various sizes, concentrations, and exposure durations. After confirming PS-MP ingestion by S. feltiae using fluorescent dyes, we found that the PS-MPs reduced the survival, reproduction, and pathogenicity of the tested EPNs, with effects intensifying for smaller PS-MPs (0.1-1 µm) at higher concentrations (105 µg/L). Furthermore, exposure to PS-MPs triggered oxidative stress in S. feltiae, leading to increased reactive oxygen species levels, compromised mitochondrial membrane potential, and increased antioxidative enzyme activity. Furthermore, transcriptome analyses revealed PS-MP-induced suppression of mitochondrial function and oxidative phosphorylation pathways. In conclusion, we show that ingestion of PS-MPs by EPNs can compromise their fitness, due to multple toxicity effects. Our results bear far-reaching consequences, as the presence of microplastics in soil ecosystems could undermine the ecological role of EPNs in regulating pest populations.

Natural occurrence of entomopathogenic nematodes (Steinernema and Heterorhabditis) and Pristionchus nematodes in black truffle soils from Spain.

The European truffle beetle Leiodes cinnamomeus is the most important pest in black truffle (Tuber melanosporum) plantations. Current control methods against it are inefficient, so entomopathogenic nematodes (EPNs) could play an important role in their population regulation due to their efficacy against many soil-dwelling insect pests. A survey of EPNs and Pristionchus nematodes was conducted in truffle soils of Spain, considering environmental and physical-chemical soil factors. A total of 164 soil samples were collected from forests, productive plantations and null-low productive plantations, representing three distinct black truffle-growing habitat types. EPNs were isolated from seven soil samples (4.3%); four nematodes were identified as Steinernema feltiae and three as Heterorhabditis bacteriophora. Both species were sampled in three types of soil texture (loam, sandy loam or sandy clay loam), characterized by alkaline pH (7.5 to 8.5) and high organic matter (2.1-11.04%). The presence of these EPNs was influenced by habitat type and organic matter content. Pristionchus nematodes were isolated from truffle soil, around truffle fruit bodies and under the elytra of L. cinnamomeus, with Pristionchus maupasi being the most commonly identified species. No significant associations were found between environmental and soil factors and the occurrence of Pristionchus nematodes. These nematodes were found in alkaline soils (pH 7.75 to 8.7), across all seven sampled soil textures, with variable organic matter content (0.73%-5.92%). The ecological trends and the presence of Pristionchus may affect the occurrence of EPNs and their prospective use as biological control agents against L. cinnamomeus in black truffle plantations.

Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Isolates in Terms of Efficacy against Cereal Ground Beetle Zabrus tenebrioides (Coleoptera: Carabidae): Morphometry and Principal Component Analysis.

One of the most dangerous pests of cereals is Zabrus tenebrioides and, in Poland, it is becoming a serious pest. Entomopathogenic nematodes (EPNs) seem to be a very promising, biological control agent for this pest. Native EPN populations are well adapted to local environmental conditions. The current study characterized three Polish isolates of the EPN Steinernema feltiae, which differed in their effectiveness against Z. tenebrioides. In the field, isolate iso1Lon reduced the pest population by 37%, compared with 30% by isolate iso1Dan and 0% by the iso1Obl isolate; the number of plants damaged by Z. tenebrioides in the presence of the different isolates reflected the results in terms of the decrease in pest population size. After incubation in the soil for 60 days, recovered EPN juveniles of all three isolates were able to infect 93-100% of the test insects, with isolate iso1Obl again showing the lowest effectiveness. The juveniles of isolate iso1Obl were also morphometrically distinct from the other two isolates, as revealed by principal component analysis (PCA), which helped to distinguish the EPN isolates. These findings showed the value of using locally adapted isolates of EPNs; two of the three isolates randomly selected from Polish soil outperformed a commercial population of S. feltiae.

Entomopathogenic nematodes for biological control of Psylliodes chrysocephala (Coleoptera: Chrysomelidae) in oilseed rape.

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

Metabonomics reveals that entomopathogenic nematodes mediate tryptophan metabolites that kill host insects.

The entomopathogenic nematode (EPN) Steinernema feltiae, which carries the symbiotic bacterium Xenorhabdus bovienii in its gut, is an important biocontrol agent. This EPN could produce a suite of complex metabolites and toxin proteins and lead to the death of host insects within 24-48 h. However, few studies have been performed on the key biomarkers released by EPNs to kill host insects. The objective of this study was to examine what substances produced by EPNs cause the death of host insects. We found that all densities of nematode suspensions exhibited insecticidal activities after hemocoelic injection into Galleria mellonella larvae. EPN infection 9 h later led to immunosuppression by activating insect esterase activity, but eventually, the host insect darkened and died. Before insect immunity was activated, we applied a high-resolution mass spectrometry-based metabolomics approach to determine the hemolymph of the wax moth G. mellonella infected by EPNs. The results indicated that the tryptophan (Trp) pathway of G. mellonella was significantly activated, and the contents of kynurenine (Kyn) and 3-hydroxyanthranilic acid (3-HAA) were markedly increased. Additionally, 3-HAA was highly toxic to G. mellonella and resulted in corrected mortalities of 62.50%. Tryptophan metabolites produced by EPNs are a potential marker to kill insects, opening up a novel line of inquiry into exploring the infestation mechanism of EPNs.

Insecticidal activities of the local entomopathogenic nematodes and cell-free supernatants from their symbiotic bacteria against the larvae of fall webworm, Hyphantriacunea.

The fall webworm (FWW), Hyphantria cunea Drury (Lepidoptera: Erebidae), is an invasive and polyphagous insect pest of many economically important crops such as hazelnuts, apple, and mulberry. Recently, there have been an increasing number of reports about the damaging activities of FWW from hazelnut growing areas of Turkey indicating that currently existing control methods fail to satisfy the expectations of growers. Entomopathogenic nematodes (EPNs) in the Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) families and the symbiotic bacteria they carry in their intestine have a great potential for the management of many agriculturally important pests. In this study, the symbiotic bacteria of local EPN species (Heterorhabditis bacteriophora AVB-15, Steinernema feltiae KCS-4S, and Steinernema bicornotum MGZ-4S) recovered from the central Anatolia region was characterized using recA gene region as Photorhabdus luminescens, Xenorhabdus bovienii and Xenorhabdus budapestensis. The contact (25, 50, 100, 200 IJs/Petri) and oral efficacies of the infective juveniles (IJs) (25, 50, 100, 200 IJs/leaf) of these EPN isolates determined on 3rd/4th instar larvae, and cell-free supernatants from the identified symbiotic bacteria were evaluated separately on the 3rd and 4th larval instars of FWW in Petri dish environment under laboratory conditions (25 ± 1 °C, 60% of RH). In the Petri dish bioassays of EPN species, the most pathogenic isolate at the 1st DAT and 4th DAT was S. feltiae which caused 50% mortality at the highest concentration (200 IJs/Petri) and the highest mortality rate (97.5%) were achieved at 4th DAT by H. bacteriophora AVB-15 isolate. Surprisingly, the mortality rates were generally higher at the lowest concentrations and 82.5% mortality were reached 4th DAT by S. bicornotum at the lowest concentration (25 IJs/leaf) in the leaf bioassays. Mortality rates were higher in both Petri dish and filter paper efficacies of cell-free supernatants at the 2nd DAT and the highest mortality (87.5%) was reached in the contact efficacy studies when applied X. bovienii KCS-4S strain. The results suggest that the tested EPN species and CFSs have good potential for biological control of the larvae of FWW and can contribute to the IPM programs of FWW. However, the efficacy of both IJs of EPNs and CFSs of their symbiotic bacteria on larvae of FWW requires further studies to verify their efficiency in the field.

Potent Ant Deterrents Emitted from Nematode-Infected Insect Cadavers.

Most known species of entomopathogenic nematodes (EPNs) are generalist obligate parasites of insects. They kill their hosts within days after infection and mortality is mainly caused by toxins produced by bacteria that co-infect the hosts and serve as food for the nematodes. EPNs can infect a very broad spectrum of insects and these insects can therefore be expected to have evolved strategies to avoid infection. Indeed, ants are known to avoid feeding on EPN-infected insect cadavers, most likely because they are repelled by semiochemicals that emanate from the cadavers. The source and nature of these repellents are so far unknown. In a series of behavioral and chemical analytical experiments we identified hexadecanal and 2-heptadecanone as two compounds that are emitted by insect larva that are infected by the EPN Steinernema feltiae, but not by uninfected larvae. When spiking honey water with the two semiochemicals, they were confirmed to be highly deterrent to the ant Lasius niger. The environmentally benign hexadecanal and 2-heptadecanone could be employed to ward off ants and possibly other pests. Additional experiments are needed to fully determine their application potential.

Microbial Control Agents for Fungus Gnats (Diptera: Sciaridae: Lycoriella) Affecting the Production of Oyster Mushrooms, Pleurotus spp.

Infestations of fungus gnats (Diptera: Sciaridae) can reduce the production of oyster mushrooms (Pleurotus spp.) grown as food crops within controlled environments. The objectives of this study were to assess the efficacy of Bacillus thuringiensis var. israelensis (Bti) and Steinernema feltiae against fungus gnat larvae. A bioassay was developed, whereby pasteurized straw was inoculated with Pleurotus columbinus and treated with Bti (Gnatrol®), S. feltiae (Nemashield®), or water. Fungus gnats (Lycoriella sp.) were released into each bioassay container for ovipositing onto the straw, thereby exposing the F1 larvae to treated or untreated substrate. Sticky cards within the containers entrapped fungus gnats emerging from the substrate as an indicator of larval survivorship. Following three bioassays, fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the water control. Three additional bioassays using Pleurotus ostreatus also demonstrated that fewer fungus gnats emerged from straw treated with Bti compared to S. feltiae and the untreated control. Steinernema feltiae was generally ineffective. Monitoring substrate weight in the bioassay containers over time indicated that Bti and S. feltiae did not impede colonization by P. ostreatus. Incorporating Bti into straw substrate is a promising approach for managing fungus gnats infesting Pleurotus spp.

Comparison of Biological Activity of Field Isolates of Steinernema feltiae with a Commercial S. feltiae Biopesticide Product.

Insect trap studies were carried out to determine the presence of entomopathogenic nematodes (EPN) from the family Steinernematidae in the soils of Poland and to compare the biological activities of field nematode isolates with nematodes from commercial biopesticide. The fauna of these organisms in central Poland is poorly studied in both taxonomic and biological terms. Tilled soils representative of this region were sampled from cultivated fields. EPN were isolated from soil samples under laboratory conditions and identified using a key for species identification and molecular analysis. Basic morphometric parameters of infective juveniles and adult males of the first generation were determined. The research showed that males and infective juveniles Steinernema feltiae from Loniów were the largest. The smallest infective juveniles were found in the isolate from Oblasy, and the smallest males in the isolate from Danków. In Poland, new field isolates showed close genetic similarity to other S. feltiae isolates. The research showed that the field isolates from Poland had greater infectivity and rate of reproduction compared with nematodes from the commercial biopesticide. The findings indicate the potential use of field S. feltiae isolates from Poland (iso1Lon, iso1Dan and iso1Obl) to develop new biopesticide products.

Field Efficacy of Steinernema sp. (Rhabditida: Steinernematidae) on the Colorado Potato Beetle Overwintering Generation.

Colorado potato beetle (CPB) is an economic pest of potato that has developed resistance to all classes of chemical insecticides, thus requiring alternative control measures. As a potential solution, entomopathogenic nematodes (EPNs) have proven effective in suppressing this pest, but their efficacy against overwintering generations of CPB in Croatia has not been sufficiently researched. The aim of this two-year (2018-2019) field study was to determine the efficacy of Steinernema feltiae and Steinernema carpocapsae applied to overwintering CPB adults. EPNs were applied at three doses (7.5 mil./10 m2, 5.0 mil./10 m2 (the recommended dose) and 2.5 mil./10 m2) by watering the soil where the adults were overwintering. The first-year results were satisfactory for both EPNs: the efficacy of S. feltiae ranged from 79.03% to 100.00%, while the efficacy of S. carpocapsae ranged from 77.32% to 96.22%. In the second year, the highest efficacy (69.57%) was obtained using the recommended dose of S. feltiae. Although the results are not consistent across the two years of our study and suggest further research, they indicate that EPNs have great potential in controlling overwintering CPB generations to reduce first generation abundance and damage, and also to prevent the spread of new generations to surrounding potato growing areas.

Efficacy and residual activity of commercially available entomopathogenic nematode strains for Mediterranean fruit fly control and their ability to infect infested fruits.

BACKGROUND: Entomopathogenic nematodes (EPN) show potential in controlling larvae of the Mediterranean fruit fly (medfly) Ceratitis capitate, but previous studies mainly concern species and strains that are not commercially available. The use of EPN for control of Mediterranean fruit fly is further hampered by the cost of using nematodes. In this study, the efficacy and residual activity of commercial strains of three EPN species, Steinernema carpοcapsae, S. feltiae and Heterοrhabditis bacteriοphοra medfly) C. capitata, in the soil substrate and inside fruits were evaluated. RESULTS: Suspensions of these species were applied at a dose of 1.5 mi m-2 on a soil substrate wherein medfly larvae were added sequentially for a period of 4 weeks post application at 20 °C. S. feltiae provided the highest suppression up to 50% as assessed by adult medfly emergence because it had the highest immediate activity and long residual activity. Furthermore, S. feltiae, and to a lesser degree S. carpocapsae, were able to move and infect medfly larvae inside infested apples and oranges left in the surface of the substrate wherein EPN were applied, reducing significantly adult medfly emergence (60-78%). CONCLUSION: These results support the efficacy and feasibility of applying a single, relatively low dose of S. feltiae in autumn, off-season, targeting overwintering medfly larvae with the scope of reducing the number of adult medflies emerging later in the new season.

Tolerance of the mealworm beetle, Tenebrio molitor, to an entomopathogenic nematode, Steinernema feltiae, at two infection foci, the intestine and the hemocoel.

An entomopathogenic nematode, Steinernema feltiae K1, exhibits pathogenicity in various insect hosts, however, its virulence among the target insect species varies. Specifically, a coleopteran insect, Tenebrio molitor, is less susceptible to S. feltiae than are lepidopteran insects. We analyzed the low virulence of S. feltiae against T. molitor sequentially, in entering the gut lumen and penetrating the hemocoel, and in hemocoelic immune defenses by comparing the responses to those of a lepidopteran insect, Spodoptera exigua. Infective juveniles (IJs) of S. feltiae exhibited higher virulence and produced more progeny IJs in S. exigua than in T. molitor. The difference in IJ behavior was observed in the IJ invasion rate (IJs in gut lumen/IJs treated) after treatment, in which a lower rate was observed in T. molitor (20.4%) than in S. exigua (55.5%). Also, a lower hemocoelic penetration rate of IJs (IJs in hemocoel/IJs in gut) was observed in T. molitor (54%) than in S. exigua (74%) 24 h after feeding treatment. To investigate the immune defense in the hemocoel, insect hemolymph samples were incubated with IJs. The encapsulation behavior and phenoloxidase activity was higher in T. molitor hemolymph than in S. exigua hemolymph, which resulted in a significantly higher nematicidal activity in S. exigua. The humoral immune responses against S. feltiae were also different between the two species. The expression of two antimicrobial peptides, cecropin and attacin 1, was much higher in T. molitor. Furthermore, eicosanoid biosynthetic activity against S. feltiae was different in the two host species; sPLA2 activity was highly inducible in T. molitor but not in S. exigua. These results suggest that variability of the immune defense in the target insects, as well as in the invasion and penetration rates of IJs to the hemocoel, plays a crucial role in determining the insecticidal virulence of S. feltiae.

Integrated Management of Chive Gnats (Bradysia odoriphaga Yang & Zhang) in Chives Using Entomopathogenic Nematodes and Low-Toxicity Insecticides.

Bradysia odoriphaga is a major pest that causes damage to chive production, and which has developed resistance to highly toxic chemical insecticides. Entomopathogenic nematodes (EPN) show a high potential for B. odoriphaga control. This study aimed to develop an effective management method against B. odoriphaga larvae, using EPN with low-toxicity insecticides. Fourteen selected insecticides had no significant effects on the survival and infectivity of Steinernema feltiae SN and Heterorhabditis indica LN2. Synergistic interactions were observed for imidacloprid and osthole with S. feltiae SN against B. odoriphaga larvae. Steinernema feltiae SN was more effective than H. indica LN2 against B. odoriphaga at 15 and 20 °C, and the addition of imidacloprid at 1/10 recommended concentration (RC) significantly increased the efficacy of S. feltiae SN. The year-round occurrence of the B. odoriphaga larvae in chive fields treated by EPN and imidacloprid at 1/10 RC was studied. Results showed that the application of EPN with imidacloprid at 1/10 RC successfully suppressed larval populations of B. odoriphaga in chive fields, thus significantly increasing the yield of chive. The practical method of applying EPN-imidacloprid combinations provided a cost-effective and environmental safety strategy for controlling B. odoriphaga larvae in chive production, which can reduce the usage of toxic chemical insecticides.

Scavenging behavior and interspecific competition decrease offspring fitness of the entomopathogenic nematode Steinernema feltiae.

Entomopathogenic nematodes (EPNs) are well-studied biocontrol agents of soil-dwelling arthropod pests. The insecticidal efficiency of EPNs is modulated by food web dynamics. EPNs can reproduce in freeze-killed insect larvae, even in competition with free-living bacterivorous nematodes (FLBNs) in the genus Oscheius. The objective of this study was to assess the efficiency of EPNs as scavengers when competing with free-living saprophagous nematodes and fungi, and to determine the possible impact on subsequent EPN offspring fitness. Live and freeze-killed larvae of Galleria mellonella were used to evaluate the reproduction rate and progeny fitness of two EPN species, Heterorhabditis bacteriophora and Steinernema feltiae, applied individually or combined with the FLBN species Oscheius onirici or Pristionchus maupasi, or Aspergillus flavus, an opportunistic saprophytic fungus. We hypothesized that (1) EPN scavenging behaviors previously observed (for H. megidis and S. kraussei) apply to other EPN species, (2) infective juveniles (IJs) emerging from freeze-killed larvae will display reduced pathogenicity and reproduction, and (3) fitness reduction will be amplified by exposure to other organisms competing for the resources. The reproduction rate of S. feltiae was lower in freeze-killed larvae than in larvae infected and killed by the nematode, whereas H. bacteriophora failed to reproduce as a scavenger. The S. feltiae F1 IJs that emerged from freeze-killed larvae exhibited lower pathogenicity rates than IJs resulting from entomopathogenic activity, and also lower reproductive rates if they experienced high FLBN competitive pressure during development. This study illustrates that scavenging is a suboptimal alternative pathway for EPNs, especially in the face of scavenger competition, even though it provides a means for some EPN species to complete their life-cycle.

Pheromone extracts act as boosters for entomopathogenic nematodes efficacy.

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Host Immunosuppression Induced by Steinernema feltiae, an Entomopathogenic Nematode, through Inhibition of Eicosanoid Biosynthesis.

Steinernema feltiae K1 (Filipjev) (Nematode: Steinernematidae), an entomopathogenic nematode, was isolated and identified based on its morphological and molecular diagnostic characteristics. Its infective juveniles (IJs) were highly pathogenic to three lepidopteran (LC50 = 23.7-25.0 IJs/larva) and one coleopteran (LC50 = 39.3 IJs/larva) insect species. Infected larvae of the diamondback moth, Plutella xylostella (L.) (Insecta: Lepidoptera), exhibited significant reduction in phospholipase A2 (PLA2) activity in their plasma. The decrease of PLA2 activity was followed by significant septicemia of the larvae infected with S. feltiae. Insecticidal activity induced by S. feltiae was explained by significant immunosuppression in cellular immune responses measured by hemocyte nodule formation and total hemocyte count (THC). Although S. feltiae infection suppressed nodule formation and THC in the larvae, an addition of arachidonic acid (AA, a catalytic product of PLA2) rescued these larvae from fatal immunosuppression. In contrast, an addition of dexamethasone (a specific PLA2 inhibitor) enhanced the nematode's pathogenicity in a dose-dependent manner. To discriminate the immunosuppressive activity of a symbiotic bacterium (Xenorhabdus bovienii (Proteobacteria: Enterobacterales)) from the nematode, kanamycin was applied to after nematode infection. It significantly inhibited the bacterial growth in the hemolymph. Compared to nematode treatment alone, the addition of antibiotics to nematode infection partially rescued the immunosuppression measured by phenol oxidase activity. Consequently, treatment with antibiotics significantly rescued the larvae from the insecticidal activity of S. feltiae. These results suggest that immunosuppression induced by infection of S. feltiae depends on its symbiotic bacteria by inhibiting eicosanoid biosynthesis, resulting in significant insect mortality. However, the addition of antibiotics or AA could not completely rescue the virulence of the nematode, suggesting that the nematode itself also plays a role in its insecticidal activity.

Nematobacterial Complexes and Insect Hosts: Different Weapons for the Same War.

Entomopathogenic nematodes (EPNs) are widely used as biological control agents against insect pests, the efficacy of these organisms strongly depends on the balance between the parasitic strategies and the immune response of the host. This review summarizes roles and relationships between insect hosts and two well-known EPN species, Steinernema feltiae and Steinernema carpocapsae and outlines the main mechanisms of immune recognition and defense of insects. Analyzing information and findings about these EPNs, it is clear that these two species use shared immunosuppression strategies, mainly mediated by their symbiotic bacteria, but there are differences in both the mechanism of evasion and interference of the two nematodes with the insect host immune pathways. Based on published data, S. feltiae takes advantage of the cross reaction between its body surface and some host functional proteins, to inhibit defensive processes; otherwise, secretion/excretion products from S. carpocapsae seem to be the main nematode components responsible for the host immunosuppression.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.

First Report of Entomopathogenic Nematode Steinernema Feltiae (Rhabditida: Steinernematidae) from Croatia.

A survey of entomopathogenic nematodes was conducted in Croatia between 2016 and 2017. The steinernematids were recovered in two out of 100 soil samples from agricultural land characterized as loamy soils with acidic reaction. Molecular and morphological identification was used to distinguish the nematodes. The isolates were identified as two different strains conspecific with Steinernema feltiae. The variations in morphometrical characteristics of infective juveniles (IJs) and males were observed among Croatian strains and with the original description. The analysis of ITS region revealed the greatest similarity of Croatian strains with Slovenian B30 and English A2 strains, which together comprised a monophyletic group in evolutionary analysis. This is the first record of steinernematids, namely S. feltiae in Croatia.